Human cells and other eukaryotes are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses “sorting signals” which are amino acid motifs located within proteins, to target proteins to particular cellular organelles.
One type of sorting signal, called signal sequence, signal peptide, or leader sequence, directs one type of proteins to an organelle called endoplasmic reticulum (ER). The ER separates the membrane-bounded proteins from all other types of proteins. Once localized to ER, both groups of proteins can be further directed to another organelle called Golgi apparatus. Here, the Golgi distributes the proteins to vesicles, including secretory vesicles, cell membranes, lysosomes, and other organelles.
Proteins targeted to the ER by a signal sequence can be released into the extracellular space as secreted proteins. For example, vesicles containing secreted proteins can fuse with the cell membranes and release their contents into the extracellular space—a process called exocytosis. Exocytosis can occur autonomously or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membranes can also be secreted into the extracellular space by proteolytic cleavage of a “linker” which holds the proteins to the membrane.
Prostate cancer is the most frequently diagnosed cancer and second leading cause of cancer death in men. 45,000 men die annually of this disease. The chance for a man to develop invasive prostate cancer during his lifetime is 1 in 6. At the age of 50, a man has a greater than 40% chance of developing prostate cancer and nearly a 3% chance of dying from this disease. Although some advances in the treatment of locally confined tumors have been achieved, prostate cancer is incurable once it has metastasized. Patients with metastatic prostate cancer are treated with hormonal ablation therapy, but with only short-term success. Eventually, these patients develop an androgen-refractory state leading to disease progression and death.
A common and fundamental problem in the treatment of prostate cancer is the absence of reliable diagnostic and prognostic markers capable of accurately detecting early-stage localized tumors and/or predicting disease susceptibility and progression. Early detection and diagnosis of prostate cancer currently relies on prostate specific antigen (PSA) assays, digital rectal examination (DRE), transrectal ultrasonography (TRUS), and transrectal needle biopsy (TRNB). Serum PSA assays in combination with DRE represent the leading diagnostic approach at present. However, this diagnostic approach has severe limitations which have fueled intensive research into finding better diagnostic markers of this disease. A number of markers have been identified, but only PSA is in widespread clinical use. However, ideal prostate tumor markers have been extremely elusive and no marker has yet been proved reliable for predicting progression of the disease. Thus, there is a need for more reliable and informative diagnostic and prognostic methods in the treatment of prostate cancer.
In addition, there is also great interest in identifying prostate-specific proteins that could be appropriate as therapeutic targets, as there is no effective treatment for patients who develop recurrent disease or who have been diagnosed with metastatic prostate cancer. Although hormone ablation therapy can palliate these patients, the majority inevitably progress to develop incurable, androgen-independent prostate cancer (Lalani et al., 1997, Cancer Metastasis Rev. 16: 29-66).
PSA is a 33 kD glycoprotein synthesized in the epithelial cells of the prostate gland. It is a secreted serine protease of the kallikrein family. The mature form of PSA takes isoleucine as the N-terminal and has 237 amino acid residues with a molecular mass of 28,400 D.
PSA is the most widely used tumor marker for screening, diagnosing, and monitoring prostate cancer today. In particular, serum PSA immunoassays are in widespread clinical use. Recently, a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for PSA mRNA in serum has been developed. However, PSA is not a prostate cancer-specific marker, since elevated levels of PSA are detectable in a large percentage of patients with BPH and prostatitis (25-86%) (Gao et al., 1997, Prostate 31: 264-281), as well as in other nonmalignant diseases and in some normal men, which is a factor significantly limits the diagnostic specificity of this marker. For example, elevations in serum PSA of between 4 to 10 ng/ml are observed in BPH, and even higher values are observed in prostatitis, particularly in acute prostatitis. BPH is an extremely common condition in men. Further confusing the situation is the fact that serum PSA elevation is observed without any indication of disease from DRE, and vice-versa. Moreover, it is now recognized that PSA not only presents in the prostate but also has a variety of complex biological activities (See e.g. Fortier et al., J. Natl. Cancer Inst. 1999, 91(19):1635-40).
While PSA-based assays are useful in the diagnosis of prostate disease, they are not sufficiently specific to distinguish the benign prostate hyperplasia (BPH) from prostate cancer (PCa). Several different approaches have been taken to improve the specificity of PSA-based assays. For example, recently, it has been found that the elevated levels of PSA (free PSA) inactive and non-complexed with alpha1-antichymotrypsin (ACT) in the serum of men with prostate cancer have been correlated with benign prostatic disease. However, none of these methodologies have been able to reproducibly distinguish benign from malignant prostate disease. In addition, PSA diagnostics has sensitivity of 57-79% (Cupp & Osterling, 1993, Mayo Clin Proc 68:297-306), and thus a significant population of men with the prostate cancer will be missed diagnosis.
Prostate-Specific Membrane Antigen (PSMA) is a recently described cell surface marker of prostate cancer which has been evaluated in various studies for its usage as a diagnostic and therapeutic marker. PSMA expression is largely restricted to prostate tissues, but detectable levels of PSMA mRNA have been observed in brain, salivary gland, small intestine, and renal cell carcinoma (Israeli et al., 1993, Cancer Res 53: 227-230). PSMA protein is highly expressed in most primary and metastatic prostate cancers, but is also expressed in most intraepithelial neoplasia specimens (Gao et al., supra). Preliminary results using an Indium-111 labeled, anti-PSMA monoclonal antibody to image recurrent prostate cancer show some promise (Sodee et al., 1996, Clin Nuc Med 21: 759-766). PSMA is a hormone dependent antigen requiring the functional androgen receptor. Since not all the prostate cancer cells express androgen receptor, the clinical utility of PSMA as a therapeutic target is inherently limited. Clinical trials designed to examine the effectiveness of PSMA immunotherapy are also underway.
Prostate Stem Cell Antigen (VISTA) is another recently described cell surface marker of prostate cancer (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735-1740). PSCA expression has been shown to be predominantly prostate specific and widely highly expressed across all stages of prostate cancer, including high differentiated prostatic intraepithelial neoplasia (PIN), androgen-dependent and androgen-independent prostate tumors. The PSCA gene has been mapped to chromosome 8q24.2, more than 80% of prostate cancers have a region of allelic. PSCA shows promise as a diagnostic and therapeutic target in view of its cell surface localization, prostate specificity, and upregulated expression in prostate cancer cells.
Progress in the identification of specific markers is slow due to lack of experimental animal model systems that recapitulate clinical prostate cancer. Attempted solutions to this problem have included the generation of prostate cancer cell lines (Horoszewicz et al., 1983, Cancer Res. 43, 1809) and prostate cancer xenografts (Pretlow et al., 1991, Cancer Res. 51, 3814; van Weerden et al., 1996, Am. J. Pathol. 149, 1055; Klein et al., 1997, Nature Med. 3, 402). However, these approaches only gain limited success. For example, xenografts have generally produced low long-term survival rates. In addition, none of the most widely used human prostate cancer cell lines—PC-3, DU-145, and LNCaP—can reproducibly give rise to osteoblastic lesions typical of prostate cancer. A further limitation of the DU-145 and PC-3 cell lines is that these cells do not express prostate specific antigen (PSA) or androgen receptor (AR) (Kaighn et al., 1979, Invest. Urol. 17: 16-23; Gleave et al., 1992, Cancer Res. 52: 1598-1605), questioning their relevance to clinical prostate cancer. The LNCaP cell line is androgen responsive and expresses PSA, but contains a mutation in the androgen receptor which alters the ligand specificity.
Recently, a series of prostate cancer xenografts (derived from patient tumors) demonstrating genetic and phenotypic characteristics similar to the human clinical situation have been described (Klein et al., 1997, Nature Med. 3: 402). These LAPC (Los Angeles Prostate Cancer) xenografts have survived more than one year in severe combined immune deficient (SCID) mice. The LAPC-4 xenograft model system has the capacity to mimic the transition from androgen dependence to androgen independence and the progress of metastatic lesions (Klein et al., 1997, supra). LAPC-4 tumors regress in male mice after castration, but re-grow within 2-3 months as androgen independent tumors. Both androgen dependent (AD) and androgen independent (AI) LAPC-4 xenograft tumors express equal levels of the prostate specific markers PSA, PSMA and PSCA (prostate stem cell antigen), which are identified using representational difference analysis of cDNAs derived from the AD and AI variants of the LAPC-4 xenograft tumors.
In one of the earliest studies on free PSA isolated from seminal plasma, internal cleavage sites at Arg85, Lys145, and Lys182 (mistakenly identified as Lys185) were observed (Watt, K. W. K., et al., Proc Natl Acad Sci USA, 83: 3166-3170, 1986). Subsequent studies have focused largely on the predominant cleavage site at Lys145 (present in 30-40% of the PSA). The presence of Lys145 cleavage is correlated with the inactivation of PSA and attributed to a random physiological cleavage, which occurs sometimes after PSA expression. The minor levels of cleavages at Arg85 and Lys182 largely ignored have also been observed (Zhang, W. M., et al., Clin Chem, 41: 1567-1573, 1995).
PSA has already been isolated from BPH tissue nodules in order to determine whether this form of PSA is different from seminal plasma PSA. BPH nodules comprise a mixture of stromal components and tightly packed epithelial ductal cells, and are visible by either macroscopic examination or low power microscopy of stained prostate tissue sections. The development of BPH nodules is highly correlated with increased prostate volume. The biochemical changes associated with nodular development may therefore play a role in the overall enlargement of the prostate, and in the clinical symptoms associated with BPH. PSA from BPH nodules has been found to contain a higher percentage of internal cleavages at Ile1, His54, Phe57, and Lys146 than seminal plasma PSA. These cleavages are thought to account for the lower enzymatic activity of PSA from BPH nodules as compared to that of the seminal plasma PSA (Price, H., et al., Hum Pathol, 21: 578-585, 1990.).